TLR-4在大鼠慢性高眼压模型视网膜中的表达

Expresstion of the TOLL-like receptor 4 in rat retina with chronic ocular hypertention

】背景TLR-4是一种天然免疫受体，在免疫性中枢神经系统损伤的修复中发挥重要作用，但在青光眼视神经损伤中的具体作用机制不详。目的从基因和蛋白质水平研究正常大鼠及慢性高眼压模型大鼠视网膜组织中TLR4的表达情况，探讨高眼压视网膜神经节细胞（RGCs）损伤的免疫机制及TLR-4在RGCs修复中的可能作用。方法选择d～5周龄的雄性清洁级纯种SD大鼠150只，用烧烙3条巩膜静脉联合术中应用丝裂霉素的方法建立大鼠慢性高眼压模型。Tono—penⅡ型笔式眼压计测量造模前、造模后2h，1、3、7、14、28、56d大鼠眼压。上述各时间点分别取5只高眼压组及空白对照组大鼠视网膜行免疫组织化学染色，观察视网膜TLR4蛋白的表达情况。应用逆转录聚合酶链反应（RT．PCR）法及Westernblot法检测各组视网膜组织中TLR-4mRNA及蛋白质的表达。结果实验组手术后眼压明显升高，与空白对照组比较差异有统计学意义（P〈0．01）。免疫组织化学检测结果显示造模后早期TLR--4在视网膜组织中的表达开始增加，表达量均高于空白对照组（P〈O．05～0．01）；RT—PCR检测表明，大鼠慢性高眼压模型眼造模后各时间点TLR-4mRNA在视网膜的表达量明显高于空白对照组（P〈0．05～0．01）；WesternNot检测显示TLR4在造模后早期视网膜组织中即有表达，结果显示组间差异有统计学意义（P〈0．05～0．01）。结论TLR-4在大鼠慢性高眼压模型眼视网膜中的表达明显上调，提示其在大鼠慢性高眼压视神经退行性病变中发挥免疫调节作用。

Background TLR-4 is a natural immunity receptors in immunity,and it plays an important role in the repair of central nervous system damage. But its effect in glaucoma optic nerve injury is unclear. Objective This study was to investigate the expression of TLR-4 in retina with high intraoeular pressure （IOP） in genetic and protein level and therefore explore the mechanism of TLR-4 on retinal ganglion cells （RGCs） injury. Methods Chronic ocular hypertension models were established in the right eyes of 150 clean purebred Sprague-Dawley rats by cauterizing the 3 sallow sclera veins. IOP was measured before and after 2 h, 1 day,3,7,14,28,56 days after operation by PEN ]I TONO-type pen tonometer. The expression of TLR-4 protein in rat retina was detected by immunohistoehemistry and Western blot, and expression of TLR-4 mRNA was assayed by real time-PCR. This experimental procedure followed the Statement of Association for Research in Vision and Ophthalmology. Results The IOP was elevated in various time points after operation in experimental group, showing significant differences in comparison with control group （P〈0.01）. The immunohistochemistry revealed that the expression of TLR-4 protein in rat retina with chronic hypertension in 2 h, 1 day, 3,7,14,28,56 days after operation with the high Az98 values in comparison with control eyes（P〈0. 05-0. 01 ）. Increased levels of TLR-4 mRNA in rat retinas were detected by RT- PCR in high lOP eyes compared with control eyes in all time points after operation,presenting statistically significant differences between two groups（P〈0. 05-0. 01 ）. Western blot detection displayed the high expression of TLR-4 in retina in high IOP eyes early after operation with statistically significant results between model group and control group （ P〈0. 05-0. 01 ）. Conclusion TLR-4 is up-regulated in rat retina with chronic high IOP, suggesting that TLR--4 plays an immunoregulatory effect in glaucomatous eye.